The biological activity of proteins often depends on conformation, where a certain tertiary or quaternary structure is needed for activity. Expression of recombinant proteins in a biologically-active conformation, for use as a therapeutic agent or in screening and diagnostic methods, is a significant technical challenge.
One approach to preparing soluble forms of the extracellular domain of transmembrane proteins is to delete the transmembrane and intracytoplasmic domains while retaining or adding an appropriate signal peptide to enable secretion of the soluble form of the protein (Smith et al., Science, 238:1704 (1987); Treiger et al., J. Immunol. 136:4099 (1986)). Another approach is to express the soluble protein as a fusion protein by joining the extracellular domain of the protein to an immunoglobulin heavy chain constant region (Fanslow et al., J. Immunol. 149:65 (1992)). However, these approaches often do not achieve, or permit the attainment of, the proper tertiary or quaternary structure required for maximal biological activity. Even if the protein is active, it may be poorly expressed or unstable. Thus, there remains a need in the art to prepare recombinant proteins that are biologically active and stable.
One set of proteins of particular interest are mature transforming growth factor-β (TGF-β) and corresponding receptors, which are involved in normal physiological processes including the regulation of cell growth, differentiation, and immune responses. TGF-β mediates signaling by binding to and complexing three types of cell surface receptors known as the TGF-βtype I (TβRI), type II (TβRII) and type III (TβRIII) receptors (Massague, J., Annu. Rev. Biochem. 67:753-791 (1998)). In the absence of ligand, both the type I and the type II receptors can form homodimers (Gilboa, L., et al., J. Cell Biol. 140:767-777 (1998)). However, these ligand-independent receptor dimers are not active due to a negative regulatory effect exerted by their extracellular domains (Zhu, H. J. and Sizeland, A. M., J. Biol. Chem. 274:29220-29227 (1999)). At least three TGF-β isoforms (TGF-β1, -β2 and -β3) are present in mammalian cells. The TGF-β1 and TGF-β3 ligand isoforms, which have a high affinity for the type II receptor extracellular domain, promote the formation of a signaling competent complex by simultaneously binding to two type II receptor extracellular domains (Letourneur, O. et al., Biochem. Biophys. Res. Commun. 224:709-716 (1996); Hart, P. J. et al., Nat. Struct. Biol. 9:203-208 (2002)). This binding event is thought to re-orient the type II receptors at the cell surface (Zhu, H. J. and Sizeland, A. M., J. Biol. Chem. 274:11773-11781 (1999)), allowing for the recruitment of two type I receptors in a signaling competent manner (Yamashita, H. et al., J. Biol. Chem. 269:20172-20178 (1994)). The type II receptor kinases can then transphosphorylate the cytoplasmic domains of the type I receptor within the complex. The signal is then translocated to the nucleus by a cascade of events involving primarily members of the Smad family (Attisano, L. and Wrana, J. L., Cytokine Growth Factor Rev. 7:327-339 (1996); Attisano, L. and Wrana, J. L., Curr. Opin. Cell Biol. 10:188-194 (1998); Massague, J., Nat. Rev. Mol. Cell Biol. 1:169-178 (2000)).
TβRIII is generally thought to be an ‘accessory’ receptor whose role is to present ligand to the signaling receptors (Lopez-Casillas, F. et al., Cell 73:1435-1444 (1993)). The idea that there is a need for this type of ‘accessory’ receptor is supported by the fact that the affinity of the TGF-β2 isoform for TβRII is low relative to the other mammalian isoforms (Cheifetz, S. et al., Cell 48:409-415 (1987); Cheifetz, S., et al. J. Biol. Chem. 265:20533-20538 (1990); Segarini, P. R. et al., Mol. Endocrinol. 3:261-272 (1989)). Recent studies suggest, however, that the role of TβRIII is more complex since TβRIII is required for both TGF-β1 or -β2 promoted mesenchymal transformation during chick embryonic heart development (Brown, C. B. et al., Science 283:2080-2082 (1999)). Also, it has been shown that the TβRIII cytoplasmic domain can be phosphorylated by, and interact with, TβRII and that this interaction is necessary for the promotion of signaling (Blobe, G. C. et al., J. Biol. Chem. 276:24627-24637 (2001)). In contrast, in other cell types, TβRIII inhibits TGF-β signaling by preventing TβRI-TβRII complex formation (Eickelberg, O. et al., J. Biol. Chem. 277:823-829 (2002)). TβRIII is found at the cell surface in a form containing glycosaminoglycan sulfate chains, which makes it electrophoretically heterogeneous (Lopez-Casillas, F. et al., Cell 67:785-795 (1991)). Two independent TGF-β binding domains were identified within the TβRIII ectodomain by mutational analysis (Fukushima, D. et al., J. Biol. Chem. 268:22710-22715 (1993); Pepin, M. C. et al., Proc. Natl. Acad. Sci. U.S.A, 91:6997-7001 (1994)). In agreement with this, the soluble ectodomain of TβRIII was shown to be able to bind to two TGF-β molecules simultaneously (De Crescenzo, G. et al., J. Biol. Chem. 276:29632-29643 (2001)).
TGF-β overexpression has been shown to play a key role in several human disorders including fibrotic diseases which are characterized by an abnormal accumulation of extracellular matrix (Border, W. A. and Noble, N. A., Am. J. Kidney Dis. 22:105-113 (1993)). It also plays a role in cancer, where TGF-β appears to play a significant role as a tumor suppressor since mutations or deletions in the genes for Smad signaling proteins and TβRII are observed in human tumors (Massague, J. et al., Cell 103:295-309 (2000)). On the other hand, there is strong evidence that, in the later stages of tumor progression, TGF-β promotes metastasis (Wakefield, L. M. and Roberts, A. B., Curr. Opin. Genet. Dev. 12:22-29 (2002)). Accordingly, it would be desirable to provide TGF-β receptors, or domains of these receptors, in their biologically-active form suitable for use as a therapeutic agent, or for use in screening and diagnostic assays. More generally, it would be desirable to provide a receptor domain of any selected protein in soluble form and in a biologically active conformation for use as a therapeutic agent and for use in various screening and diagnostic assays, and preferably in a cell free assay.